The overall goals of the project are to determine the molecular mechanisms that mediate the anti-angiogenic activity of thrombospondin-1 (TSP-1). These mechanisms include the binding of TSP-1 to the CD36 multiprotein complex and other receptors and the induction of endothelial cell apoptosis. Specific focus during the proposed period of support will be placed in the following areas: Specific Aim 1: Characterization of the CD36 multiprotein complex involved in mediating TSP-1 effects on endothelial cells. We hypothesize that (1) CD36 associates with specific integrins and tetraspanins in the endothelial cell membrane, (2) tetraspanins and integrins collaborate with CD36 to form a TSP-1 binding site, and (3) TSP-1 drives the recruitment of specific proteins to these complexes. CD36-associated proteins will be identified by immunoprecipitation followed by mass spectrometry and Western blotting. Endothelial cells are solubilized in detergents with varying abilities to disrupt hydrophobic interactions as a first step in distinguishing direct and indirect associations. Co-immunolocalization studies by confocal and electron microscopy will be used to confirm the biochemical data. Expression vectors for integrins, tetraspanins and other CD36-associated proteins will be used alone or in combination to identify the proteins that are required for TSP-1 binding to CD36-transfected COS-7 cells. These expression systems will also be used to assay for direct and indirect associations between the members of the CD36-multiprotein complex. Specific Aim 2: Characterization of the molecular mechanism of thrombospondin-1 induced apoptosis. We hypothesize that TSP-1-dependent inhibition of angiogenesis is mediated via activation of death receptor-mediated apoptotic pathways, the inactivation of anti-apoptotic pathways and altered expression of apoptotic or anti-apoptotic genes. Endothelial cells from normal and TSP-1-deficient mice, human dermal microvascular endothelial cells, melanoma (B16F10) or pancreatic cancer cells (ASPC-1 and CFPAC) will be treated with TSP-1 and recombinant domains of TSP-1 (that bind to CD36 and TGFb or just CD36). We will determine the role of Fas receptor and TRAIL ligand in mediating the effect of TSP-1 using decoy receptors or dominant inhibitory mutants of the receptor in vitro, and lpr mice deficient in Fas receptor. The role of the activated FADD, caspase-8 or caspase-9 will be determined by dominant inhibitory or activating approaches. We will (1) determine the expression pattern of known apoptotic genes, (2) analyze the effects of TSP-1 on the anti-apoptotic pathways by in vivo kinase assays (for Akt activity) and transcriptional activity assays (for BCL-2 family members), (3) evaluate the ability of TSP-1 domains to induce endothelial and tumor cell apoptosis in experimental melanoma and pancreatic cancer, and (4) investigate whether or not the TSP-1-deficient and TSP-1, CD36 double knockout mice show a defect in the apoptosis of their endothelial cells and other cell types.